Development of Synthetic Microenvironments for Stem Cell Growth and Differentiation
Currently, many chronic diseases and injuries do not have effective cures; millions of people suffer from disabilities while carrying on daily lives without appropriate medical assistance. Advances in human pluripotent stem cells (hPSCs) research have provided the potential hope for significant improvements of disease treatment and management. The success of stem cell-based therapy will have major impacts on the quality of life of people with chronic health problems such as cancer, cardiovascular diseases, and neurodegenerative disorders (e.g. Alzheimer’s and Parkinson’s diseases). The California Institute of Regenerative Medicine was established to develop such novel cell-based therapies to treat disorders that are presently incurable. HPSCs process the enormous potential to be directed to all cell types in the human body as the “raw material” for many cell-based therapies. The realization of the full potential of hPSCs in regenerative medicine requires, among other things, the establishments of well-defined culture conditions for their growth and differentiation and cost-effective protocols for their expansion. In this grant application, we propose a series of experiments to develop a novel technology platform using cost-effective and well-defined synthetic matrices to expand and differentiate hPSCs. The results will provide critical information and protocols for hPSC researchers aiming at developing cures for the diseases mentioned above.
Due to the high cost and limited range of testing capability, previous studies on factors affecting stem cell growth have focused on only one or a few elements of the cellular microenvironment, e.g., individual extracellular matrix components or growth factors. In addition, most protocols for hPSC culture use non-human products such as animal supporting materials and recombinant proteins isolated from bacterial culture, which represent potential complications for clinical usage. Our proposed research will develop a high-throughput cellular microarray screening tool that incorporates synthetic materials, including polymers and peptides, to select the optimal matrix for supporting self-renewal and differentiation of hPSCs. This tool and technology will allow the concomitant screening of the effects of thousands of conditions on growth, maintenance and differentiation of hPSCs with a cost-effective approach. The results from our studies will provide fully defined and optimized culture conditions for the expansion and differentiation of hPSCs without exposure to animal-derived products.
In summary, we will develop a comprehensive approach to elucidate the responses of hPSCs to microenvironmental factors in a combinatorial and systematic manner. Application of this novel and powerful technology will lead to the definition of the optimal synehtic matrices for the control of hPSC growth and differentiation and the production of hPSCs without contamination by non-human products.
Many California families have suffered or will suffer from diseases and injuries that do not have an effective cure. Current medical treatments can manage, but cannot cure, diseases and injuries such as cancer, heart diseases, spinal cord injuries, Alzheimer’s, and Parkinson’s disease. Recent advances in human pluripotent stem cells (hPSCs) studies have provided the opportunity to develop novel strategies involving cell-replacement therapies to overcome the inadequacy of conventional drug-based treatments. However, cell-replacement therapies require sufficiently large numbers of clinically viable hPSCs that have been thoroughly tested and characterized. To address this critical need, we have developed a comprehensive and cost-effective technology for high-throughput screening of native matrix proteins that regulate hPSC growth and differentiation. The aim of our current research is to apply this high-throughput technology to synthetic polymers and peptides to establish cost-effective protocols for efficient and precise controls of hPSCs growth and differentiation.
Our technology will allow the screening of thousands of well-defined material properties to identify the optimal synthetic matrix for stem cell growth and differentiation. In contrast to current bio-reagents used in hPSC studies, our focus on synthetic materials will enable the development of cost-effective, scalable, robust platform for generating clinically viable hPSCs or hPSC-derived cells. Our technology, which we will make freely available to the biomedical community, will also benefit many other lines of scientific inquiries, such as defining growth conditions of rare adult stem cell populations. Thus, our proposed research is fundamental to the clinical applications of hPSCs in regenerative medicine and has broad benefits to a wide spectrum of scientific interests.
Our research will not only benefit the health of the people, but also the economy of California by enhancing and generating local businesses. With this project, we will be able to hire researchers to conduct and manage the proposed research. In addition, the outcome of this project will lead to the development of a biotechnology platform that can provide great benefits to the advancement of California biotechnology industry. The patents, royalties and licensing fees that result from advances in the proposed research will provide tax revenues to California. Thus, this proposed research project provides not only the essential foundation for the scientific progresses in stem cell biology and regenerative medicine to improve health and quality of life, but also potential technology advancement and financial gain for the people in the State of California.
The goal of the project is to develop well-defined synthetic matrices composed of polymers and peptides that provide the optimum cell-matrix interactions for hPSC proliferation and differentiation by using a microarray technology recently developed in our laboratory. We have selected PMVE-alt-MA and PSS-hydorgel (coplolymer) as the candidates for supporting hPSC expansion; other polymers will also be tested. A platform for peptide array has been established for the future peptide screening for hPSC expansion and differentiation. We identified poly(allylamine hydrochloride) as a polymer that supports differentiated hNPC self renewal; other polymers and peptides will be tested for supporting hPSC differentiation. The time line remains the same.
The goal of the project is to develop well-defined synthetic matrices composed of polymers and peptides to support stem cell growth and differentiation. Over the past year, we have used our array-based, high-throughput approach for the systematic investigation of the effects of polymers and peptides on human pluripotent stem cell (hPSCs) maintenance and differentiation. We have also extended our studies to neural progenitor cells (NPCs) derived from hPSCs because they offer a unique model system to study neural development and are a possible source of cells to treat a variety of neurodegenerative disorders. Current practices for deriving, expanding, and differentiating NPCs generally utilize empirically determined combinations of reagents, which are expensive, difficult to isolate, subject to batch-to-batch variations, and unsuitable for cell-based therapies. In contrast, synthetic polymers that are inexpensive and easy to fabricate represent a reliable alternative for in vitro NPC expansion.
We found that a combination of physical cues modulate protein adsorption to our previously developed hydrogels as well as the signaling properties of these proteins, which in turn directly affect human pluripotent stem cell (hPSCs) maintenance and differentiation. We identified of a number of candidate polymers that support short-term NPC attachment, growth, and maintenance of pluripotency. Furthermore, we are starting to test large polymer coated slides to investigate the scalability of the polymers’ physiochemical properties and biological performance. Our data shows that the optimum polymer composition was able to support the attachment and growth of NPCs and that they retained the ability to differentiate to cells with neuronal-like morphologies.
In addition, we also diversified our microarray technology to use a library of synthetic peptides that are designed to have similar functionalities to those of extracellular matrix proteins. These peptides retain the functional properties of proteins, are more stable and versatile, and are relatively inexpensive. Preliminary data demonstrate that hPSCs are able to adhere to these peptides in a dose dependent manner.
Previously established extracellular matrix protein (ECMP) and growth factor (GF) array technology are currently testing optimal ECMP-GF combinations for their ability to maintain and stabilize a definitive endoderm population over multiple passages. These experiments are an extension of several recent publications from our group. Although these studies do not involve synthetic polymers or peptides, they provide important proof-of-principle that this screening platform can be applied to the differentiation and expansion of definitive endoderm.
Advances in human pluripotent stem cells (hPSCs) research have provided the potential hope for significant improvements of disease treatment and management. The success of stem cell-based therapy will have major impacts on the quality of life of people with chronic health problems such as cancer, cardiovascular diseases, and neurodegenerative disorders. The realization of the full potential of hPSCs in regenerative medicine requires, among other things, the establishments of well-defined culture conditions for their growth and differentiation and cost-effective protocols for their expansion. In this grant application, we propose a series of experiments to develop a novel technology platform using cost-effective and well-defined synthetic matrices to expand and differentiate hPSCs. During the funding period, we developed a comprehensive approach to elucidate the responses of hPSCs, as well as neuronal progenitor cells (NPCs) to microenvironmental factors in a combinatorial and systematic manner. Application of this novel and powerful technology will lead to the definition of the optimal synehtic matrices for the control of hPSC growth and differentiation and the production of hPSCs and NPCs without contamination by non-human products.
The potentials of using hPSCs in regenerative medicine are enormous. However, there is a critical need to overcome the hurdles of using hPSCs in regenerative medicine, such as the high cost for hPSC expansion/differentiation, the lack of identification of optimal microenvironment, and the potential contamination with animal products. The central goal of the proposed studies is to develop a synthetic matrix-based culture system for promoting self-renewal and lineage-specific differentiation. Furthermore, due to the limitation of current pharmacological therapeutic strategies, hPSC-derived neural progenitor cells (hNPCs), a multipotent cell population that is capable of near indefinite expansion and subsequent differentiation into the various cell types that comprise the central neuron system (CNS), could provide an unlimited source of cells for such cell-based therapies. We have developed a high-throughput array technology that allows simultaneous screening of the effects of a large number of materials/conditions on the behavior of cells. In this research project, we applied this technology to facilitate the precise and efficient development of optimal matrices comprising of synthetic polymers for hPSC growth and neuronal differentiation, with the goal of establishing an effective and clinically applicable procedure for the scaled-up production of hPSCs and hPSC-derived hNPCs.
In sum, our study combined the expertise of an interdisciplinary team including bioengineering, material sciences, and stem cell biology to develop a novel cellular microarray technology that allowed us to develop optimal synthetic polymers for large-scale expansion of hPSCs and hNPCs. All of these are achieved in a defined environment free of animal products and in a cost-effective manner, which will enable the application of such technology for translational therapy using hPSCs and hNPCs.